DNA replication in metazoan cells has been one of the most important areas of research in biology. Even though great progress has been made in characterizing the proteins involved in this process and the regulatory circuitry evolved to ensure the fidelity of replication, there are still many fundamentally important questions that have not yet been fully understood about these specialized regions in the eukaryotic genome. One of these is the selection and maintenance of active replication start sites in complex initiation zones found at most metazoan replication origins. In this project, we propose to apply two single molecule techniques, atomic force microscopy and DNA mapping with molecular combing, to directly examine the details of DNA replication at the B-globin locus from individual human cells as a model system. Specifically, we wish (1) to determine whether multiple start sites in the initiation zone at the B-globin locus could be utilized simultaneously in a given S-phase, thus, to determine if a mechanism similar to "origin interference" found in yeast should also apply to other higher eukaryotes as a general principle of replication initiation; (2) to discover whether a specific start site used in one cell cycle could be "memorized" in subsequent S-phases, thus, to determine if an active start site could be maintained by an epigenetic mechanism; (3) to further correlate these studies with the distribution of selected chromosomal proteins in the vicinity of these loci using ChIP and optical mapping on combed DNA at high spatial resolution, thus, to determine if any of the known chromatin structural proteins can be directly linked to the selection and maintenance of active replication start sites. With these studies, not only do we wish to reveal critical insights on the nature and utility of complex replication origins, but also to further establish these single molecule methods for the study of DNA replication in individual eukaryotic cells. When such methods are combined with other established techniques, we will be able to further unravel various molecular determinants and the fundamental principles of genomic replication that should also have practical implications.